Intestinal epithelial cells participate in immune regulation and mucosal integrity. Tight junctions (TJs) constitute continuous circumferential seals around cells and serve as a protective barrier, preventing solutes and water from passing freely through the paracellular pathway. Tight junctions can be altered by various pathogens, as well as by their toxins. These effects may result from direct modification of TJ proteins such as occludin, claudin, and Zonula occludens-1 (ZO-1), or by alteration of the perijunctional actomyosin ring (Berkes et al., “Intestinal Epithelial Responses to Enteric Pathogens: Effects on the Tight Junction Barrier, Ion Transport, and Inflammation,” Gut 52: 439-451 (2003); Landau, “Epithelial Paracellular Proteins in Health and Disease,” Curr Opin Nephrol Hypertens 15: 425-429 (2006); Sousa et al., “Microbial Strategies to Target, Cross or Disrupt Epithelia,” Curr Opin Cell Biol 17:489-498 (2005)).
Salmonella enterica serovar Typhimurium is a major cause of human gastroenteritis. Infection of polarized epithelial cell monolayers by S. Typhimurium disrupts TJ structure and function (Finlay et al., “Salmonella Interactions with Polarized Human Intestinal Caco-2 Epithelial Cells,” J Infect Dis 162:1096-1106 (1990); Jepson et al., “Rapid Disruption of Epithelial Barrier Function by Salmonella Typhimurium is Associated with Structural Modification of Intercellular Junctions,” Infect Immun 63:356-359 (1995); Jepson et al., “Localization of Dysfunctional Tight Junctions in Salmonella enterica serovar Typhimurium-infected Epithelial Layers,” Infect Immun 68:7202-7208 (2000); Tafazoli et al., “Disruption of Epithelial Barrier Integrity by Salmonella enterica serovar Typhimurium Requires Geranylgeranylated Proteins,” Infect Immun 71:872-881 (2003)). TJ disruption is dependent on the type III secretory system (TTSS) of Salmonella. TTSS is a needle-like protein transport device used by Gram-negative pathogenic bacteria. It allows bacteria to inject virulence effectors into eukaryotic host cells (Galan, “Salmonella Interactions with Host Cells: Type III Secretion at Work,” Annu Rev Cell Dev Biol 17:53-86 (2001)). TTSS is encoded by the Salmonella pathogenicity island 1 (SPI-1) (Galan, “Interaction of Salmonella with Host Cells through the Centisome 63 Type III Secretion System,” Curr Opin Microbiol 2:46-50 (1999)). A recent study indicated that SopB, SopE, SopE2, and SpiA are the TTSS secreted SPI-1 effectors responsible for the disruption of TJ structure and function (Boyle et al., “Salmonella enterica serovar Typhimurium Effectors SopB, SopE, SopE2 and SipA Disrupt Tight Junction Structure and Function,” Cell Microbiol 8:1946-1957 (2006)). The specific bacterial effectors responsible for the regulation of TJs, however, remain to be identified. The majority of published studies regarding Salmonella and TJ have utilized in vitro cultured epithelial models. The physiological consequences of Salmonella-effector-induced alteration of TJ function need to be addressed in vivo using animal models.
AvrA is a newly described bacterial effector transported into the host cell by the TTSS of Salmonella (Hardt et al., “A Secreted Salmonella Protein with Homology to an Avirulence Determinant of Plant Pathogenic Bacteria,” Proc Natl Acad Sci USA 94:9887-9892 (1997)). It also belongs to the SPI-1 (Hardt et al., “A Secreted Salmonella Protein with Homology to an Avirulence Determinant of Plant Pathogenic Bacteria,” Proc Natl Acad Sci USA 94:9887-9892 (1997)). The SPI-1 effectors are responsible for early inflammation in the mouse model of S. Typhimurium-induced enterocolitis (Hapfelmeier et al., “Role of the Salmonella Pathogenicity Island 1 Effector Proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium Colitis in Streptomycin-pretreated Mice,” Infect Immun 72:795-809 (2004); Barthel et al., “Pretreatment of Mice with Streptomycin Provides a Salmonella enterica serovar Typhimurium Colitis Model that Allows Analysis of Both Pathogen and Host,” Infect Immun 71:2839-2858 (2003)). AvrA protein from Salmonella Typhimurium inhibits activation of the proinflammatory NF-κB transcription factor in cultured human epithelial cells (Collier-Hyams et al., “Cutting Edge: Salmonella AvrA Effector Inhibits the Key Proinflammatory, Anti-apoptotic NF-kappa B Pathway,” J Immunol 169:2846-2850 (2002)). Based on the sequence alignment, AvrA belongs to the cysteine protease family (Orth et al., “Disruption of Signaling by Yersinia effector YopJ, a Ubiquitin-like Protein Protease,” Science 290:1594-1597 (2000)). Representative AvrA members include the adenovirus-like proteases (human adenovirus type 2, fowl adenovirus 8, Hemorrhagic enteritis virus), YopJ (Yersinia outer protein J), and AvrBsT. The catalytic triad for the cysteine protease is present in all AvrA family members (Orth et al., “Disruption of Signaling by Yersinia effector YopJ, a Ubiquitin-like Protein Protease,” Science 290:1594-1597 (2000); Orth et al., “Inhibition of the Mitogen-activated Protein Kinase Kinase Superfamily by a Yersinia Effector,” Science 285:1920-1923 (1999)). Further studies demonstrated that expression of a mutant AvrA protein with a single amino acid residue transition (AvrA/C186A) in a putative catalytic cysteine of this enzyme did not inhibit TNFα-stimulated induction of the reporter (Collier-Hyams et al., “Cutting Edge: Salmonella AvrA Effector Inhibits the Key Proinflammatory, Anti-apoptotic NF-kappa B Pathway,” J Immunol 169:2846-2850 (2002)). It was recently demonstrated that AvrA has deubiquitinase activity which removes ubiquitins from ub-IκBα, thus inhibiting NF-κB activity (Ye et al., “Salmonella effector AvrA Regulation of Colonic Epithelial Cell Inflammation by Deubiquitination,” Am J Pathol 171:882-892 (2007)). AvrA C186A mutant protein had reduced deubiquitinase activity as evidenced by cleaving less ubiquitin moieties from IκBα (Ye et al., “Salmonella effector AvrA Regulation of Colonic Epithelial Cell Inflammation by Deubiquitination,” Am J Pathol 171:882-892 (2007)). This data further supports the hypothesis that AvrA protein has protease activity which attenuates the proinflammatory NF-κB pathway.
The AvrA gene is present in 80% of Salmonella enterica serovars (Streckel et al., “Expression Profiles of Effector Proteins SopB, SopD1, SopE1, and AvrA Differ with Systemic, Enteric, and Epidemic Strains of Salmonella enterica,” Mol Nutr Food Res 48:496-503 (2004)). The protein expression of AvrA differs strikingly between bacterial strains in systemic disease and in enteritis, which is localized to the intestine (Streckel et al., “Expression Profiles of Effector Proteins SopB, SopD1, SopE1, and AvrA Differ with Systemic, Enteric, and Epidemic Strains of Salmonella enterica,” Mol Nutr Food Res 48:496-503 (2004)). AvrA protein was not expressed in strains related to systemic disease, but was conditionally (pH below 6.0) expressed in the enteritis-related strains. In addition, S. enterica strains from systemic infections could be characterized by their strong SopB and SopE1 expression and by the absence of SopD1 and AvrA proteins (Streckel et al., “Expression Profiles of Effector Proteins SopB, SopD1, SopE1, and AvrA Differ with Systemic, Enteric, and Epidemic Strains of Salmonella enterica,” Mol Nutr Food Res 48:496-503 (2004)). Four phenotypic classes of S. enterica have been identified under defined standard culture conditions: strains with a constitutive synthesis of AvrA; strains with an acid induction of AvrA; strains with silent avrA genes; and a fourth class without AvrA gene (Ben-Barak et al., “The Expression of the Virulence-associated Effector Protein Gene avrA is Dependent on a Salmonella enterica-specific Regulatory Function,” Int J Med Microbiol 296:25-38 (2006)). Taken together, AvrA protein expression is very different from the other Salmonella effectors such as SopB, SopD, and SopE (Ben-Barak et al., “The Expression of the Virulence-associated Effector Protein Gene avrA is Dependent on a Salmonella enterica-specific Regulatory Function,” Int J Med Microbiol 296:25-38 (2006)). Although it is premature to claim a correlation of AvrA with the clinical and epidemiological potency of Salmonellae, current studies indicate that a fine-tuning of AvrA expression takes place during the pathogenesis of Salmonella infection.
Unlike SopB and SopD, AvrA does not increase physiologic fluid secretion into infected calf ileal loops (Zhang et al., “The Salmonella enterica serotype typhimurium Effector Proteins SipA, SopA, SopB, SopD, and SopE2 Act in Concert to Induce Diarrhea in Calves,” Infect Immun 70:3843-3855 (2002); Schesser et al., “The Salmonella YopJ-homologue AvrA does not Possess YopJ-like Activity,” Microb Pathog 28:59-70 (2000)). However, the role of AvrA expression on the tight junction structure and function of the intestinal epithelial cells in both in vitro and in vivo models is unexplored.
The present invention overcomes these and other deficiencies in the art, and identifies a therapeutic mechanism for treatment of Inflammatory Bowel Disease (“IBD”), Celiac Disease, and other inflammatory conditions of the intestine.